The two nuclear genes, CYC1 and CYC7, encoding the mitochondrial proteins iso-1-cytochrome c and iso-2-cytochrome c, respectively, in the yeast Saccharomyces cerevisiae comprise one of the most thoroughly studied gene- protein systems of eukaryotes. All steps of CYC1 gene expression, have been systematically examined, including transcription, translation, co- translational and post-translational modification, mitochondrial import, heme attachment, and enzymatic functions. In addition, methods have been developed for altering the CYC1 and CYC7 genes by transforming yeast directly with synthetic oligonucleotides. This system, along with other pertinent genes, will be used to investigate various aspects of transcription termination, translation, co- and post-translational modification, mitochondrial import, and the degradation of mitochondrial protein components. 3' End forming signals will be investigated by systematically altering the 3' region of the CYC1 gene. The function of SUT1, a gene involved in proper 3' end formation, will be examined. This system will be used to investigate role of mRNA structures on the translational context effect. The sequences required for trimethylation of Lys77 will be investigated with altered forms of iso- 1-cytochrome c having amino acid replacements. The enzymes and the N-terminal amino acid sequences required for N-terminal acetylation will be systematically investigated. Mitochondrial import of cytochrome c will be investigated, including import signals and the role of heme. Major emphasis will be on the characterization of the various degradation systems that act on cytochrome c and other mitochondrial proteins, including: (i) the presumably cytosolic protease that specifically degrades apo-iso-1- cytochrome c, but not apo-iso-2-cytochrome c, and that may be encoded by CYC12; (ii) the ATh-dependent mitochondrial protease that presumably act generally on all mitochondrial proteins including holo-cytochrome c; (iii) a newly uncovered degradation system that acts on certain labile forms of iso- 1 -cytochrome c and iso-2-cytochrome c, and that may involve the SUD1 gene. For these studies, we have at our disposable a wide range of altered iso-l-cytochromes c with known thermodynamic properties, including not only common labile forms that are rapidly degraded, but also those that are more stable than the wildtype form and that are more resistant to degradation in vivo. We will also investigate the newly uncovered phenomenon of "mutual protection", in which cytochrome a-a3 protects the degradation of certain labile forms of the iso-cytochromes c, and in which cytochrome c prevents the degradation of cytochrome a-a3.